Results

Range and habitat

We detected L. nigriventer at the known and newly identified sites within the Hula Nature Reserve, and at another location, about 1 km southeast of the reserve borders near the small village Yesod HaMa’ala (Fig. 1 C–F). In total, we observed 64 adult females, 42 adult males, 29 juveniles (Fig. 2) and 40 tadpoles. Of these, six middle-sized to large individuals (SVL 33.8–76.8 mm) were discovered within the reserve, while 112 medium to large-sized individuals (SVL 43.0–128.4 mm), 19 small individuals (SVL 16.2–30.1 mm; Fig. 2 M) as well as tadpoles at Gosner stages 25–34 were recorded at Yesod HaMa’ala. Based on our specimen records we estimate L. nigriventer to occupy an area of at least 6.5 km², but the number of active reproductive sites is uncertain.

The species was found to exploit different kinds of terrestrial and aquatic habitats:

(1) In the Hula Nature Reserve all individuals were discovered in terrestrial habitats. As a former part of the Hula marshes and lake, the organic soil at this site is peaty, damp and loose, and covered by a ca. 20–30 cm layer of humid decomposed leaf litter. Most individuals (including 9 of the 14 individuals found prior to our surveys) were found beneath this layer within a dense thicket of blackberry (Rubus sanguineus Frivaldszky, 1835), reeds (Phragmites australis (Cavanilles) Trinius Ex Steudel, 1840) and occasionally fig trees (Ficus carica Linnaeus, 1753). The majority of the reserve’s water bodies are lentic and about 20–30% of the permanently flooded area is covered by dense stands of reeds and Papyrus sedge (Cyperus papyrus Linnaeus, 1753). It is, however, uncertain which of the water bodies of the Hula Nature Reserve are used by L. nigriventer as no individuals were found here in their aquatic habitat, and no eggs or larvae were detected.

(2) At the site near Yesod HaMa’ala, all individuals were found in the water or at the slopes of a ~600 m long ditch. This ditch has a permanent source of water from a small spring and dense vegetation both in and next to the water. The water is very slowly flowing and depth is substantial (up to ca. 150 cm) but starting at depths of ca. 10–100 cm. A deep layer of mud covers the bottom of the ditch and aquatic vegetation covers much of the water’s surface (comprising dense P. australis growths, water lettuce (Pistia stratiotes Linnaeus, 1753), and duckweed, Lemna minor Linnaeus, 1753). At the edges of the ditch, the mineral soil is compressed, sandy and in the dry season less damp than in the reserve. Individuals in terrestrial habitat were detected either beneath dried or half-dried grass tufts, or in natural cavities or small burrows at the water edge dug by semi-terrestrial freshwater crabs (Potamon potamios (Olivier, 1804)) or small mammals. The Latonia individuals encountered at this site displayed a strikingly high percentage of injury. Of the 112 medium to large-sized frogs (> 42 mm), 28% had old or recent minor injuries mainly on the hind limbs (Fig. 3), while such injuries were not detected in juvenile frogs or in individuals found in the Hula Nature Reserve. However, the ditch is less frequently visited by migrating birds or larger mammals and has fewer fish species than other, major water bodies in the Hula Nature Reserve (see S2 in the Supplement).

Activity patterns and movement

All tadpoles were caught in shallow parts of the Yesod HaMa’ala ditch. Metamorphosed individuals were observed either within humid leaf litter in the close vicinity to a perennial water body, directly at the water’s edge or in the water. The majority of the individuals discovered during winter months – especially during the first long-term field trip from November 2013 through March 2014 – were land-dwelling juveniles of snout-vent lengths of ~20–30 mm. Adults were mostly detected in the water from February through September, which may correspond to the breeding period (Fig. 4).

Fig. 4. Number of individuals of Latonia nigriventer captured across months. All individuals below 66 mm were considered juveniles, males and females were distinguished on the basis of their foot webbing and the presence of nuptial pads in males.

In general, the frogs appeared to be mainly nocturnal and could frequently be observed in the water after nightfall. Individuals were found solitarily and were not observed in aggregations.

Upon capture, terrestrial individuals first froze in their movement and then tried to escape forward by either slowly jumping or walking. Walking individuals retracted their eyes. Whenever the density of the soil allowed, they forced their way underground head first with retracted eyes by strongly pushing back all limbs in a walking manner. The increased secretion of viscous skin mucus further facilitated the movement through soil or vegetation. Individuals observed at night in the water were usually submerged with exception of the rostral portion of the head (Fig. 2 N). Upon disturbance, they immediately tried to retreat into the water and escape head first into dense thickets of aquatic plants and roots. They were more easily scared (e.g. by our electric torches) than P. bedriagae or H. savignyi. While being handled, adults of both sexes uttered release calls that were similar to the presumed advertisement calls described below but less regular and less intense.

For the frogs for which displacement was recorded by capture-recapture, the distances moved were < 20 m (N = 10), > 20 m < 100 m (N = 4) and ≤ 100 m (N = 5) (Table 1). Three radio-tracked frogs moved no more than 2 m over periods of 5, 5 and 18 days, respectively.

Adult morphology

Latonia nigriventer is a rather inconspicuously coloured, robust frog reaching large sizes of 69.0–128.4 mm in females and 66.6–121.4 mm in males. In the observed individuals, webbing was strongly developed suggesting a substantial aquatic adaptation, and hind limbs were comparatively short. The head was rather flat and the iris heart-shaped. A distinct transversal dermal fold was present in the neck. The colour pattern was similar in all individuals despite differences in distinctness and contrast (Fig. 2). An incomplete mid-dorsal band of the lighter colour was almost always visible in the posterior part of the dorsum. The venter was black to grey with a distinct pattern of white spots which corresponded to raised tubercles in adults. For a detailed morphological description of adults, see Appendix 1; for data and measurements of the holotype, see Appendix 2.

From mid-February until mid-September, i.e. during the presumed breeding season, we observed distinct dark nuptial pads and more or less distinct black, keratinised excrescences on the thorax, ventral part of arms and thighs, plantar surfaces as well as on the outer edge of the webbing in males. In single cases, excrescences were also present on dorsal surface of feet. In females, such structures were distinct only on plantar surfaces and to some degree on the webbing edge, and in very rare cases, single excrescences were seen on the thoracic region (Fig. 5). The excrescences on the body skin had the form of isolated spicules whereas at the webbing edges they formed dense aggregations. The smallest male with clearly developed nuptial pads had a snout-vent length of 66.6 mm, and we therefore used this value as cut-off to distinguish adults from juveniles. Based on specimens > 66 mm, we found no significant difference in the size-weight distribution of males and females (body condition calculated with the relative m­ass (Wr ) condition index; Sztatecsny and Schabetsberger, 2005) using a Mann-Whitney-U test (p = 0.615; N (female/male) = 64/44; Fig. 6), nor did we detect distinct sexual dimorphism in morphometric measurements (Table 2). Slight differences were however seen in foot webbing (see S3 in the Supplement): females had slightly weaker webbing, with a distinct broadening of the toe III webbing usually at the 3rd phalanx, and on toe IV at the 3rd–4th phalanx; whereas in males it usually was seen at the 2nd phalanx at toe III and the 3rd phalanx at toe IV. Forearms were strong in both sexes and could not be reliably used to differentiate females from males (for raw measurements see S4 in the Supplement).

Tadpole morphology

Tadpoles collected at two distinct sites in the ditch near Yesod HaMa’ala on 15 May 2015 agree with the description of Mendelssohn and Steinitz (1943) in having double keratodont rows and a network of black epidermal lines. They were small (total length 14 mm at Gosner stage 25 and 24 mm at Gosner stage 34), uniformly brown, with an unpigmented ventral side and ventral spiracle. The LTRF was 2/3(1), not considering double rows (Fig. 7). For a detailed description of the tadpoles see Appendix 3, for raw measurements see S5 in the Supplement.

The tadpoles of L. nigriventer are distinguished from those of the three other anuran species in this area (Bufotes variabilis (Pallas, 1769), H. savignyi and P. bedriagae) as well as those of Pelobates syriacus Boettger, 1889, by their medial ventral spiracle, distinct epidermal reticulations and double keratodont rows. Such biserial rows of keratodonts are a unique state for the basal anuran genera Discoglossus and Ascaphus, and are, with exception of Hoplobatrachus, not found in more advanced frogs (Grosjean et al., 2004). Dorsally, L. nigriventer tadpoles mostly resemble the ones of Bufotes in the position of their eyes and general appearance, but they lack the distinctly golden speckled venter as observed in the latter. The unpigmented venter of L. nigriventer tadpoles likewise serves as an immediate distinguishing characteristic from both Hyla and Pelophylax tadpoles that display a silvery venter.

Of the 40 tadpoles (Gosner stages roughly between 25 and 35) found on 15 May, 9 were successfully raised to post-metamorphs at the School of Marine Sciences (Ruppin Academic Center), Michmoret, where metamorphosis took place between 3–15 June 2015. Recently metamorphosed frogs had a SVL of 6–9 mm. At the end of June, all post-metamorphs were transferred to the Garden of Zoological Research at Tel Aviv University for further rearing.

Reproduction

Besides the observation of tadpoles in mid-May, no direct observation of breeding has been made. The dissection of a dead L. nigriventer female that had been overrun by a tractor in mid-January revealed several hundred greyish-black oocytes with a diameter between 1.5–2 mm. From the available data and phylogenetic relatedness it can be hypothesised that at least some aspects of the reproduction are similar to Discoglossus, which are opportunistic breeders with a short and intense inguinal amplexus during which several batches of eggs are deposited and adhere to stones, aquatic plants or the bottom of the water body. According to the observations of (i) males with distinct nuptial pads and other keratinised excrescences from February to September, (ii) tadpoles in mid-May (our observation) and August (Mendelssohn and Steinitz, 1943), and (iii) the observed weight losses in recaptured females of L. nigriventer, we hypothesise a relatively prolonged reproductive period with egg depositions potentially taking place at least from March to June, and possible from February to September. This extended reproductive season, if confirmed, would be longer than in the three sympatric amphibian species, i.e., February/March–May in B. variabilis, March/April–June in H. savignyi, and May–August/September in P. bedriagae (Degani and Mendelssohn, 1984).

Vocalisations

Similar to Discoglossus (Weber, 1974; Glaw and Vences, 1991; Vences and Glaw, 1996), the calls heard from L. nigriventer were of very low intensity, low spectral frequency, and consisting of a presumed expiratory and a presumed inspiratory note. As the release calls of handled L. nigriventer sound very similar to the recorded calls, we assume the calls to be uttered at the water surface like in Discoglossus species. While we cannot exclude that other, more intense call types can be emitted by this species, the absence of externally visible vocal sacs in males makes it likely that their vocalisations mainly serve short-distance communication. Although we could only achieve provisional recordings under suboptimal conditions in captivity, which might have distorted some of the call features, we were able to record the calls of several male L. nigriventer individuals. Air temperature during recordings varied between 13.5–18 °C and water temperature between 14–15 °C. As several males were kept together in the same aquarium, the following descriptive statistics refer to calls of various males in unknown proportions.

Calls were mostly uttered in a series and were separated from each other by short intervals of silence varying from 246–1606 ms (mean + SD: 787 + 609 ms; N = 65). Each call consisted of two notes which we assume represent sounds produced by expiration (first note) followed by inspiration of air into the lungs (second note). Both notes were spectrally structured and pulsatile, but a clear distinction and count of pulses was not possible. The two notes of one call were not separated by a silent interval or distinct decrease in amplitude. Therefore, in the spectrogram the two notes are mostly recognisable by the somewhat lower frequency and higher intensity of the second (inspiratory) note (Fig. 8). Dominant frequency peak averaged over the total call (mean + SD) was 775.5 + 80 Hz (N = 72); frequency range was roughly between 0–1500 Hz. Call duration (N = 72) ranged between 725–1212 ms, with the expiratory note being longer (671 + 115 ms) than the inspiratory note (291 + 28 ms) (Table 3).

Fig. 8. Spectrogram and oscillogram of series of A) two and B) nine presumed advertisement calls of Latonia nigriventer. Note the low frequency (< 1.5 kHz). The two notes (presumed expiratory and inspiratory) within each call are distinguishable, yet not separated by a silent interval. Sampling rate 44.1 kHz.

Table3. Call features of the presumed advertisement calls recorded from a group of several individuals of Latonia nigriventer.

The pathogen Bd was detected in 32% of the tested amphibian individuals (n = 87) from northern Israel, while none were positive for Bsal. We found Bd in two amphibian species (L. nigriventer and P. bedriagae) and in three of the seven examined locations within the Hula Valley (Hula Nature Reserve, Kiryat Shmona and Yesod HaMa’ala). Infection loads for Bd-positive individuals ranged between 1–311 genomic equivalents of zoospores per swab (Table 4).

Table 4. Number of individuals per species tested for Batrachochytrium dendrobatidis including mean (range) of genomic equivalents of zoospores per swab for positive tested specimen.

Bacterial communities of L. nigriventer were comprised of Proteobacteria (56.7%) with a high representation of Gammaproteobacteria (33.4% of the overall community), Bacteroidetes (25.3%) and Firmicutes (6.7%) (Fig. 9 A). The 20 most abundant OTUs found on the skin of L. nigriventer represented 42% of the total reads (Fig. 9 B; S6 in the Supplement). The most abundant OTU (7% of the total sequences) was assigned to an unspecified Chryseobacterium and present in 100% of the samples, although in varying abundance (< 1–24% of the reads). Comparisons based on weighted UniFrac distances did not reveal significant differences between (i) microbial communities from the ventral versus dorsal skin of L. nigriventer (PERMANOVA: N = 27; p = 0.117; Fig. 10 A), (ii) ventral surfaces of females versus males (PERMANOVA: N = 15; p = 0.646; Fig. 10 B) or (iii) ventral surfaces of Bd-positive versus Bd-negative individuals (PERMANOVA: N = 22; p = 0.283; Fig. 10 C). However, we observed a significant shift in the ventral skin microbial community over time.

Fig. 9. Relative abundances of major bacterial taxa obtained from Latonia nigriventer skin samples in Yesod HaMa’ala as identified by the SILVA 119 database. The order of the taxa in columns corresponds to that in legends (ordered alphabetically). A) Abundances of dominant bacterial phylotypes; B) abundances of the 20 most frequent bacterial OTUs. (For detailed OTU IDs see S5 in the Supplement).

Fig. 10. Principal coordinates analysis plots of weighted UniFrac distances of the microbial communities associated with Latonia nigriventer and Pelophylax bedriagae. A) Comparison of dorsal and ventral surfaces in L. nigriventer; B) comparison of ventral skin microbial communities found in female and male L. nigriventer; C) comparison of L. nigriventer individuals tested positive and negative for chytrid (ventral surfaces only); D) seasonal changes in the ventral skin microbial community associated with L. nigriventer; E) comparison of the ventral skin microbial communities found in P. bedriagae and L. nigriventer; F) Venn diagram depicting the overlap of core microbial communities as obtained from ventral skin swabs of L. nigriventer and P. bedriagae captured at the same location and day. The minimum fraction of samples an OTU must be observed in was set to 75%.

While no significant changes were observed between the ventral skin samples taken in mid-February and mid-April (N = 13; p = 0.799) or between those collected in mid-April and the end of June (N = 15; p = 0.0.093), significant differences were detected for all other time-associated comparisons: mid-February – late June (N = 15; p = 0.012); mid-February – mid-September (N = 15; p = 0.001); late June – mid-September (N = 17; p = 0.001) (Fig. 10 D).

The results obtained for the skin-associated bacterial communities of syntopic P. bedriagae were similar to those of L. nigriventer: no significant differences between ventral versus dorsal surfaces of the same individuals (N = 14; p = 0.898) nor between ventral surfaces of Bd-positive versus Bd-negative individuals (N = 22; p = 0.366).

A comparison of the ventral skin-associated communities of L. nigriventer and P. bedriagae from the same location and same time-point revealed differences between the two species (N = 17; p = 0.001; Fig. 10 E). The core bacterial communities contained 30 OTUs (88% of the core skin microbiota of L. nigriventer and 57% of that of P. bedriagae) that were present on the ventral skin of at least 75% of the individuals of both species (Fig. 10 F).

The skin secretions collected from two different individuals and examined for peptide composition had significant amounts of hydrophobic peptides recovered after C18 enrichment. We detected a number of common peptide mass signals shared by both frog individuals. The mass ranges are suggestive of possible antimicrobial peptides (Table 5; S7 in the Supplement). In a growth inhibition assay, the mixture of peptides inhibited the growth of two different Bd isolates (JEL 197 and ‘Section Line’; Fig. 11). At the highest concentration tested (500 μg/ml), Bd growth inhibition ranged from 51% to 91.5% against the Section Line isolate and 70–82% inhibition against the original type isolate JEL 197. Both isolates are among the global panzootic lineages (Schloegel et al., 2012; Piovia-Scott et al., 2015). Furthermore, the direct skin secretion solution was found to inhibit Bd by 35–36%.

Table 5. Quantity of peptides detected in the mucus of two Latonia nigriventer females.